Photocleavable protecting groups and methods for their use

ABSTRACT

Novel compounds are provided which are useful as linking groups in chemical synthesis, preferably in the solid phase synthesis of oligonucleotides and polypeptides. These compounds are generally photolabile and comprise protecting groups which can be removed by photolysis to unmask a reactive group. The protecting group has the general formula Ar—C(R 1 )(R 2 )—O—C(O)— wherein:  
     Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogous derivative thereof;  
     R 1  and R2 are independently H, optionally substituted alkyl, alkenyl or alkynyl, optionally substituted aryl or optionally substituted heteroaromatic, or a vinylogous derivative of the foregoing; and  
     X is a leaving group, a chemical fragment linked to Ar—C(R 1 )(R 2 )—O—C(O)— via a heteroatom, or a solid support; provided that when Ar is 1-pyrenyl and R 1  and R 2  are H, X is not linked to Ar—C(R 1 )(R 2 )—O—C(O)— via a nitrogen atom. Preferred embodiments are those in which Ar is a fused polycyclic aromatic hydrocarbon and in which the substituents on Ar, R 1  and R 2  are electron donating groups. A particularly preferred protecting group is the “PYMOC” protecting group, pyrenylmethyloxycarbonyl, where Ar is pyrenyl and R 1  and R 2  are H.  
     Also provided is a method of forming, from component molecules, a plurality of compounds on a support, each compound occupying a separate predefined region of the support, using the protected compounds described above.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a division of U.S. patent applicationSer. No. 09/525,045, filed on Mar. 14, 2000; which is a continuation ofU.S. patent application Ser. No. 08/812,005, filed on Mar. 5, 1997, nowU.S. Pat. No. 6,147,205; which is a continuation-in-part of U.S. patentapplication Ser. No. 08/630,148, filed Apr. 10, 1996, now U.S. Pat. No.6,022,963; which is a regular application of Provisional PatentApplication No. 60/008,684, filed Dec. 15, 1995, all of which areincorporated herein by reference.

[0002] The present invention was made with U.S. Government support underATP Grant No. 70NANB5H1031, and the government may have certain rightsin the invention.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to the area of chemical synthesis.More particularly, this invention relates to photolabile compounds,reagents for preparing the same and methods for their use asphotocleavable linkers and protecting groups, particularly in thesynthesis of high density molecular arrays on solid supports.

[0004] The use of a photolabile molecule as a linker to couple moleculesto solid supports and to facilitate the subsequent cleavage reaction hasreceived considerable attention during the last two decades. Photolysisoffers a mild method of cleavage which complements traditional acidic orbasic cleavage techniques. See, e.g., Lloyd-Williams et al. (1993)Tetrahedron 49:11065-11133. The rapidly growing field of combinatorialorganic synthesis (see, e.g., Gallop et al. (1994) J. Med. Chem.37:1233-1251; and Gordon et al. (1994) J. Med. Chem. 37:1385-1401)involving libraries of peptides and small molecules has markedly renewedinterest in the use of photolabile linkers for the release of bothligands and tagging molecules.

[0005] A variety of ortho-benzyl compounds as photolabile protectinggroups have been used in the course of optimizing the photolithographicsynthesis of both peptides (see Fodor et al. (1994) Science 251:767-773)and oligonucleotides (see Pease et al. Proc. Natl. Acad. Sci. USA91:5022-5026). See PCT patent publication Nos. WO 90/15070, WO 92/10092,and WO 94/10128; see also U.S. patent application Ser. No. 07/971,181,filed Nov. 2, 1992, and Ser. No. 08/310,510, filed Sep. 22, 1994; Holmeset al. (1994) in Peptides: Chemistry, Structure and Biology (Proceedingsof the 13th American Peptide Symposium); Hodges et al. Eds.; ESCOM:Leiden; pp. 110-12, each of these references is incorporated herein byreference for all purposes. Examples of these compounds included the6-nitroveratryl derived protecting groups, which incorporate twoadditional alkoxy groups into the benzene ring. Introduction of ana-methyl onto the benzylic carbon facilitated the photolytic cleavagewith >350 nm UV light and resulted in the formation of a nitroso-ketone.

[0006] Photocleavable protecting groups and linkers should be stable toa variety of reagents (e.g., piperidine, TFA, and the like); be rapidlycleaved under mild conditions; and not generate highly reactivebyproducts. The present invention provides such protecting groups andmethods for their use in synthesizing high density molecular arrays.

SUMMARY OF THE INVENTION

[0007] According to a first aspect of the invention, novel compounds areprovided which are useful for providing protecting groups in chemicalsynthesis, preferably in the solid phase synthesis of oligonucleotidesand polypeptides. These compounds are generally photolabile and compriseprotecting groups which can be removed by photolysis to unmask areactive group. The compounds have the general formulaAr—C(R₁)(R₂)—O—C(O)—X, wherein:

[0008] Ar is an optionally substituted fused polycyclic aryl orheteroaromatic group or a vinylogous derivative thereof;

[0009] R₁ and R₂ are independently H, optionally substituted alkyl,alkenyl or alkynyl, optionally substituted aryl or optionallysubstituted heteroaromatic, or a vinylogous derivative of the foregoing;and

[0010] X is a leaving group, a chemical fragment linked toAr—C(R₁)(R₂)—O—C(O)— via a heteroatom, or a solid support; provided thatwhen Ar is 1-pyrenyl and R₁═R₂═H, X is not linked toAr—C(R₁)(R₂)—O—C(O)— via a nitrogen atom. Preferred embodiments arethose in which Ar is a fused polycyclic aromatic hydrocarbon and inwhich the substituents on Ar, R₁ and R₂ are electron donating groups.Particularly preferred protecting groups are the “PYMOC” protectinggroup, pyrenylmethyloxycarbonyl, where Ar=1-pyrenyl and R₁═R₂═H, and the“ANMOC” protecting group, anthracenylmethyloxycarbonyl, whereAr=anthracenyl and R₁═R₂═H. Methods are provided for preparing thesecompounds

[0011] This invention also provides reagents of the molecular formulaAr—C(R₁)(R₂)—O—C(O)—X, where Ar, R₁, and R₂ have the meanings ascribedabove, for incorporating the protecting group into the molecule desiredto be protected.

[0012] Another aspect of this invention provides a method of attaching amolecule with a reactive site to a support comprising the steps of:

[0013] (a) providing a support with a reactive site;

[0014] (b) binding a molecule to the reactive site, the moleculecomprising a masked reactive site attached to a photolabile protectinggroup of the formula Ar—C(R₁)(R₂)—O—C(O)—, wherein:

[0015] Ar is an optionally substituted fused polycyclic aryl orheteroaromatic group or a vinylogously substituted derivative of theforegoing;

[0016] R₁ and R₂ are independently H, optionally substituted alkyl,alkenyl or alkynyl, or optionally substituted aryl or heteroaromaticgroup or a vinylogously substituted derivative of the foregoing;

[0017] to produce a derivatized support having immobilized thereon themolecule attached to the photolabile protecting group; and

[0018] (c) removing the photolabile protecting group to provide aderivatized support comprising the molecule with an unmasked reactivesite immobilized thereon.

[0019] A related aspect of this invention provides a method of forming,from component molecules, a plurality of compounds on a support, eachcompound occupying a separate predefined region of the support, saidmethod comprising the steps of:

[0020] (a) activating a region of the support;

[0021] (b) binding a molecule to the region, said molecule comprising amasked reactive site linked to a photolabile protecting group of theformula Ar—C(R₁)(R₂)—O—C(O)—, wherein:

[0022] Ar is an optionally substituted fused polycyclic aryl orheteroaromatic group or a vinylogously substituted derivative of theforegoing;

[0023] R₁ and R₂ are independently H, optionally substituted alkyl,alkenyl or alkynyl, or optionally substituted aryl or heteroaromaticgroup or a vinylogously substituted derivative of the foregoing;

[0024] (c) repeating steps (a) and (b) on other regions of the supportwhereby each of said other regions has bound thereto another moleculecomprising a masked reactive site linked to the photolabile protectinggroup, wherein said another molecule may be the same or different fromthat used in step (b);

[0025] (d) removing the photolabile protecting group from one of themolecules bound to one of the regions of the support to provide a regionbearing a molecule with an unmasked reactive site;

[0026] (e) binding an additional molecule to the molecule with anunmasked reactive site;

[0027] (f) repeating steps (d) and (e) on regions of the support until adesired plurality of compounds is formed from the component molecules,each compound occupying separate regions of the support.

[0028] The present invention also provides methods of performingchemical reactions on a surface, by providing at least one chemicalreactant on the surface, and applying a coating to the surface. Thecoating provides an environment that is favorable to reaction of thechemical reactant. In a more preferred aspect, the invention provides amethod of activating a functional group on a surface that is protectedwith a protecting group of the invention. The method involves applying anucleophilic coating to the surface, and exposing the surface to lightto remove the protecting group.

[0029] The methods and compositions described herein find particularutility in the synthesis of high density arrays of compounds, andparticularly nucleic acids, on solid supports.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIGS. 1 and 2 show syntheses of 5′-O-pyrenylmethyloxycarbonyl(“PYMOC”) protected deoxyribonucleoside 3′-O-cyanoethylphosphoramiditesin a suitable form for coupling to a support.

[0031]FIG. 3 shows representative Ar groups that can be present in thecompounds of this invention.

[0032]FIG. 4 illustrates fluorescent scans of substrates subjected todry front-side exposure, coated front-side exposure and wet exposure(water/MeOH in a flow cell). Two coatings (0.1% Triton X-100/H₂O and0.2% Triton X-100/50% glycerol/H₂O) were tested and the results areshown in panels A and B, respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0033] The following definitions are set forth to illustrate and definethe meaning and scope of the various terms used to describe theinvention herein.

[0034] The term “alkyl” refers to a branched or straight chain acyclic,monovalent saturated hydrocarbon radical of one to twenty carbon atoms.

[0035] The term “alkenyl” refers to an unsaturated hydrocarbon radicalwhich contains at least one carbon-carbon double bond and includesstraight chain, branched chain and cyclic radicals.

[0036] The term “alkynyl” refers to an unsaturated hydrocarbon radicalwhich contains at least one carbon-carbon triple bond and includesstraight chain, branched chain and cyclic radicals.

[0037] The term “aryl” refers to an aromatic monovalent carbocyclicradical having a single ring (e.g., phenyl) or two condensed rings(e.g., naphthyl), which can optionally be mono-, di-, ortri-substituted, independently, with alkyl, lower-alkyl, cycloalkyl,hydroxylower-alkyl, aminolower-alkyl, hydroxyl, thiol, amino, halo,nitro, lower-alkylthio, lower-alkoxy, mono-lower-alkylamino,di-lower-alkylamino, acyl, hydroxycarbonyl, lower-alkoxycarbonyl,hydroxysulfonyl, lower-alkoxysulfonyl, lower-alkylsulfonyl,lower-alkylsulfinyl, trifluoromethyl, cyano, tetrazoyl, carbamoyl,lower-alkylcarbamoyl, and di-lower-alkylcarbamoyl. Alternatively, twoadjacent positions of the aromatic ring may be substituted with amethylenedioxy or ethylenedioxy group. Typically, electron-donatingsubstituents are preferred.

[0038] The term “heteroaromatic” refers to an aromatic monovalent mono-or poly-cyclic radical having at least one heteroatom within the ring,e.g., nitrogen, oxygen or sulfur, wherein the aromatic ring canoptionally be mono-, di- or tri-substituted, independently, with alkyl,lower-alkyl, cycloalkyl, hydroxylower-alkyl, aminolower-alkyl, hydroxyl,thiol, amino, halo, nitro, lower-alkylthio, lower-alkoxy,mono-lower-alkylamino, di-lower-alkylamino, acyl, hydroxycarbonyl,lower-alkoxycarbonyl, hydroxysulfonyl, lower-alkoxysulfonyl,lower-alkylsulfonyl, lower-alkylsulfinyl, trifluoromethyl, cyano,tetrazoyl, carbamoyl, lower-alkylcarbamoyl, and di-lower-alkylcarbamoyl.For example, typical heteroaryl groups with one or more nitrogen atomsare tetrazoyl, pyridyl (e.g., 4-pyridyl, 3-pyridyl, 2-pyridyl), pyrrolyl(e.g., 2-pyrrolyl, 2-(N-alkyl)pyrrolyl), pyridazinyl, quinolyl (e.g.2-quinolyl, 3-quinolyl etc.), imidazolyl, isoquinolyl, pyrazolyl,pyrazinyl, pyrimidinyl, pyridonyl or pyridazinonyl; typical oxygenheteroaryl radicals with an oxygen atom are 2-furyl, 3-furyl orbenzofuranyl; typical sulfur heteroaryl radicals are thienyl, andbenzothienyl; typical mixed heteroatom heteroaryl radicals are furazanyland phenothiazinyl. Further the term also includes instances where aheteroatom within the ring has been oxidized, such as, for example, toform an N-oxide or sulfone.

[0039] The term “optionally substituted” refers to the presence or lackthereof of a substituent on the group being defined. When substitutionis present the group may be mono-, di- or tri-substituted,independently, with alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl,aminolower-alkyl, hydroxyl, thiol, amino, halo, nitro, lower-alkylthio,lower-alkoxy, mono-lower-alkylamino, di-lower-alkylamino, acyl,hydroxycarbonyl, lower-alkoxycarbonyl, hydroxysulfonyl,lower-alkoxysulfonyl, lower-alkylsulfonyl, lower-alkylsulfinyl,trifluoromethyl, cyano, tetrazoyl, carbamoyl, lower-alkylcarbamoyl, anddi-lower-alkylcarbamoyl. Typically, electron-donating substituents suchas alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl, aminolower-alkyl,hydroxyl, thiol, amino, halo, lower-alkylthio, lower-alkoxy,mono-lower-alkylamino and di-lower-alkylamino are preferred.

[0040] The term “electron donating group” refers to a radical group thathas a lesser affinity for electrons than a hydrogen atom would if itoccupied the same position in the molecule. For example, typicalelectron donating groups are hydroxy, alkoxy (e.g. methoxy), amino,alkylamino and dialkylamin0.

[0041] The term “leaving group” means a group capable of being displacedby a nucleophile in a chemical reaction, for example halo, nitrophenoxy,pentafluorophenoxy, alkyl sulfonates (e.g., methanesulfonate), arylsulfonates, phosphates, sulfonic acid, sulfonic acid salts, and thelike.

[0042] “Activating group” refers to those groups which, when attached toa particular functional group or reactive site, render that site morereactive toward covalent bond formation with a second functional groupor reactive site. For example, the group of activating groups which canbe used in the place of a hydroxyl group include —O(CO)Cl; —OCH₂Cl;—O(CO)OAr, where Ar is an aromatic group, preferably, a p-nitrophenylgroup; —O(CO)(ONHS); and the like. The group of activating groups whichare useful for a carboxylic acid include simple ester groups andanhydrides. The ester groups include alkyl, aryl and alkenyl esters andin particular such groups as 4-nitrophenyl, N-hydroxylsuccinimide andpentafluorophenol. Other activating groups are known to those of skillin the art.

[0043] “Chemical library” or “array” is an intentionally createdcollection of differing molecules which can be prepared eithersynthetically or biosynthetically and screened for biological activityin a variety of different formats (e.g., libraries of soluble molecules;and libraries of compounds tethered to resin beads, silica chips, orother solid supports). The term is also intended to refer to anintentionally created collection of stereoisomers.

[0044] “Predefined region” refers to a localized area on a solid supportwhich is, was, or is intended to be used for formation of a selectedmolecule and is otherwise referred to herein in the alternative as a“selected” region. The predefined region may have any convenient shape,e.g., circular, rectangular, elliptical, wedge-shaped, etc. For the sakeof brevity herein, “predefined regions” are sometimes referred to simplyas “regions.” In some embodiments, a predefined region and, therefore,the area upon which each distinct compound is synthesized smaller thanabout 1 cm² or less than 1 mm². Within these regions, the moleculesynthesized therein is preferably synthesized in a substantially pureform. In additional embodiments, a predefined region can be achieved byphysically separating the regions (i.e., beads, resins, gels, etc.) intowells, trays, etc.

[0045] “Solid support”, “support”, and “substrate” refer to a materialor group of materials having a rigid or semi-rigid surface or surfaces.In many embodiments, at least one surface of the solid support will besubstantially flat, although in some embodiments it may be desirable tophysically separate synthesis regions for different compounds with, forexample, wells, raised regions, pins, etched trenches, or the like.According to other embodiments, the solid support(s) will take the formof beads, resins, gels, microspheres, or other geometric configurations.

[0046] Isolation and purification of the compounds and intermediatesdescribed herein can be effected, if desired, by any suitable separationor purification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography,thick-layer (preparative) chromatography, distillation, or a combinationof these procedures. Specific illustrations of suitable separation andisolation procedures can be had by reference to the exampleshereinbelow. However, other equivalent separation or isolationprocedures can, of course, also be used.

[0047] A “channel block” is a material having a plurality of grooves orrecessed regions on a surface thereof. The grooves or recessed regionsmay take on a variety of geometric configurations, including but notlimited to stripes, circles, serpentine paths, or the like. Channelblocks may be prepared in a variety of manners, including etchingsilicon blocks, molding or pressing polymers, etc.

[0048] This invention provides novel compounds which are useful forproviding protecting groups in chemical synthesis, preferably in thesolid phase synthesis of oligonucleotides and polypeptides and highdensity arrays thereof. These compounds are generally photolabile andcomprise protecting groups which can be removed by photolysis to unmaska reactive group. The compounds have the general formulaAr—C(R₁)(R₂)—O—C(O)—X, wherein:

[0049] Ar is an optionally substituted fused polycyclic aryl orheteroaromatic group or a vinylogous derivative thereof,

[0050] R₁ and R₂ are independently H, optionally substituted alkyl,alkenyl or alkynyl, optionally substituted aryl or optionallysubstituted heteroaromatic, or a vinylogous derivative of the foregoing;and

[0051] X is a leaving group, a chemical fragment linked toAr—C(R₁)(R₂)—O—C(O)— via a heteroatom, or a solid support; provided thatwhen Ar is 1-pyrenyl and R₁═R₂═H, X is not linked toAr—C(R₁)(R₂)—O—C(O)— via a nitrogen atom.

[0052] Preferred embodiments are those in which Ar is a fused polycyclicaromatic hydrocarbon and in which the substituents on Ar, R₁ and R₂ areelectron donating groups, such as alkoxy groups, particularly one ormore methoxy groups. Examples of electron donating carrying R₁ and R₂groups are methyl, substituted phenyl groups, e.g., o- orp-methoxyphenyl; 2,6-dimethoxyphenyl; 2,3-dimethoxyphenyl;3,5-dimethoxyphenyl and the like. Other R₁ and R₂ groups include9-anthracenyl or 1-pyrenyl. Particularly preferred protecting groups arethe “PYMOC” protecting group, 1-pyrenylmethyloxycarbonyl, whereAr=1-pyrenyl and R₁═R₂═H, and the “ANMOC” protecting group,anthracenylmethyloxycarbonyl, where Ar=anthracenyl (e.g. 9-anthracenyl)and R₁═R₂═H.

[0053] Representative fused polycyclic aromatic hydrocarbons includenaphthalene, phenanthrene, anthracene, benzoanthracene,dibenzoanthracene, heptalene, acenaphtbalene, acephenanthrene,triphenylene, pyrene, fluorene, phenalene, naphthacene, picene,perylene, pentaphenylene, pyranthrene, fullerenes (including C₆₀ andC₇₀), and the like. A representative vinylogously substituted derivativeof an aromatic hydrocarbon is styrene.

[0054] The invention also provides reagents of the molecular formulaAr—C(R₁)(R₂)—O—C(O)—X, where Ar, R₁, and R₂ have the meanings ascribedabove, for incorporating the protecting group into the molecule desiredto be protected. X can be any suitable leaving group such as halo,oxycarbonyl, imidazolyl, pentafluorophenoxy and the like, which iscapable of reacting with a nucleophilic group such as hydroxy, amino,alkylamino, thio and the like on the molecule being protected. Thus, thereagents comprising the protecting groups Ar—C(R₁)(R₂)—O—C(O)— disclosedherein can be used in numerous applications where protection of areactive nucleophilic group is required. Such applications include, butare not limited to polypeptide synthesis, both solid phase and solutionphase, oligo- and polysaccharide synthesis, polynucleotide synthesis,protection of nucleophilic groups in organic syntheses of potentialdrugs, etc.

[0055] The invention also provides compositions of the molecular formulaAr—C(R₁)(R₂)—O—C(O)—M, where Ar, R₁ and R₂ have the meaning outlinedabove and M is any other chemical fragment. Preferably, M will be amonomeric building block that can be used to make a macromolecule. Suchbuilding blocks include amino acids, peptides, polypeptides, nucleicacids, nucleotides, nucleosides, monosaccharides, and the like.Preferred nucleosides are ribonucleosides and deoxyribonucleosides suchas adenosine, deoxyadenosine, cytidine, deoxycytidine, thymidine,uracil, guanosine and deoxyguanosine as well as oligonucleotidesincorporating such nucleosides. Preferably, the building block is linkedto the photolabile protecting group via a hydroxy or amine group. Whennucleotide and oligonucleotide compositions are used, with theprotecting groups of this invention, the protecting groups arepreferably incorporated into the 3′-OH or the 5′-OH of the nucleoside.Other preferred compounds are protected peptides, proteins,oligonucleotides and oligodeoxyribonucleotides. Small organic molecules,proteins, hormones, antibodies and other such species havingnucleophilic reactive groups can be protected using the protectinggroups disclosed herein.

[0056] The use of nucleoside and nucleotide analogs is also contemplatedby this invention to provide oligonucleotide or oligonucleoside analogsbearing the protecting groups disclosed herein. Thus the termsnucleoside, nucleotide, deoxynucleoside and deoxynucleotide generallyinclude analogs such as those described herein. These analogs are thosemolecules having some structural features in common with a naturallyoccurring nucleoside or nucleotide such that when incorporated into anoligonucleotide or oligonucleoside sequence, they allow hybridizationwith a naturally occurring oligonucleotide sequence in solution.Typically, these analogs are derived from naturally occurringnucleosides and nucleotides by replacing and/or modifying the base, theribose or the phosphodiester moiety. The changes can be tailor made tostabilize or destabilize hybrid formation or enhance the specificity ofhybridization with a complementary nucleic acid sequence as desired.

[0057] Analogs also include protected and/or modified monomers as areconventionally used in oligonucleotide synthesis. As one of skill in theart is well aware oligonucleotide synthesis uses a variety ofbase-protected deoxynucleoside derivatives in which one or more of thenitrogens of the purine and pyrimidine moiety are protected by groupssuch as dimethoxytrityl, benzyl, tert-butyl, isobutyl and the like.Specific monomeric building blocks which are encompassed by thisinvention include base protected deoxynucleoside H-phosphonates anddeoxynucleoside phosphoramidites.

[0058] For instance, structural groups are optionally added to theribose or base of a nucleoside for incorporation into anoligonucleotide, such as a methyl, propyl or allyl group at the 2′-Oposition on the ribose, or a fluoro group which substitutes for the 2′-Ogroup, or a bromo group on the ribonucleoside base.2′-O-methyloligoribonucleotides (2′-O-MeORNs) have a higher affinity forcomplementary nucleic acids (especially RNA) than their unmodifiedcounterparts. 2′-O-MeORNA phosphoramidite monomers are availablecommercially, e.g., from Chem Genes Corp. or Glen Research, Inc.Alternatively, deazapurines and deazapyrimidines in which one or more Natoms of the purine or pyrimidine heterocyclic ring are replaced by Catoms can also be used.

[0059] The phosphodiester linkage, or “sugar-phosphate backbone” of theoligonucleotide analogue can also be substituted or modified, forinstance with methyl phosphonates or O-methyl phosphates. Anotherexample of an oligonucleotide analogue for purposes of this disclosureincludes “peptide nucleic acids” in which a polyamide backbone isattached to oligonucleotide bases, or modified oligonucleotide bases.Peptide nucleic acids which comprise a polyamide backbone and the basesfound in naturally occurring nucleosides are commercially availablefrom, e.g., Biosearch, Inc. (Bedford, Mass.).

[0060] Nucleotides with modified bases can also be used in thisinvention. Some examples of base modifications include 2-aminoadenine,5-methylcytosine, 5-(propyn-1-yl)cytosine, 5-(propyn-1-yl)uracil,5-bromouracil, and 5-bromocytosine which can be incorporated intooligonucleotides in order to increase binding affinity for complementarynucleic acids. Groups can also be linked to various positions on thenucleoside sugar ring or on the purine or pyrimidine rings which maystabilize the duplex by electrostatic interactions with the negativelycharged phosphate backbone, or through hydrogen bonding interactions inthe major and minor groves. For example, adenosine and guanosinenucleotides can be substituted at the N² position with an imidazolylpropyl group, increasing duplex stability. Universal base analogues suchas 3-nitropyrrole and 5-nitroindole can also be included. A variety ofmodified oligonucleotides and oligonucleotide analogs suitable for usein this invention are described in, e.g., “Antisense Research andApplications”, S. T. Crooke and B. LeBleu (eds.) (CRC Press, 1993) and“Carbohydrate Modifications in Antisense Research” in ACS Symp. Ser.#580, Y. S. Sanghvi and P. D. Cook (eds.) ACS, Washington, D.C. 1994).

[0061] Compounds of this invention can be prepared by carbonylating anaromatic carbinol of the general formula Ar—C(R₁)(R₂)—OH with acarbonylation reagent such as for example, phosgene (COCl₂),carbonyldiimidazole or pentafluorophenoxy chloroformate and the like toprovide Ar—C(R₁)(R₂)—O—C(O)—X where X is a leaving group derived fromthe carbonylating reagent (Cl, if phosgene was used, pentafluorophenoxy,if pentafluorophenoxy chloroformate was used, etc.). This intermediate,Ar—C(R₁)(R₂)—O—C(O)—X is then reacted with a molecule M carrying anucleophilic group whose protection is desired to yield a protectedbuilding block Ar—C(R₁)(R₂)—O—C(O)-M. Representative aromatic carbinolsare pyrenemethanol, naphthalenemethanol, anthracenemethanol,perylenemethanol and the like. Such aromatic carbinols are availablefrom commercial suppliers such as Aldrich Chemical Co., Milwuakee, Wis.Alternatively, they may also be obtained from precursor aromatichydrocarbons by acylation under Friedel-Crafts conditions with acidchlorides and anhydrides and subsequent reduction of the carbonyl groupthus added to a carbinol.

[0062] Alternatively, one may first carbonylate the group on themolecule being protected with a carbonylation reagent, such as onedescribed above, and subsequently displace the leaving group X thusinserted with the hydroxyl group of the aromatic carbinol. In eitherprocedure, one frequently uses a base such as triethylamine ordiisopropylethylamine and the like to facilitate the displacement of theleaving group.

[0063] One of skill in the art will recognize that the protecting groupsdisclosed herein can also be attached to species not traditionallyconsidered as “molecules”. Therefore, compositions such as solidsurfaces (e.g., paper, nitrocellulose, glass, polystyrene, silicon,modified silicon, GaAs, silica and the like), gels (e.g., agarose,sepharose, polyacrylamide and the like) to which the protecting groupsdisclosed herein are attached are also contemplated by this invention.

[0064] The protecting groups of this invention are typically removed byphotolysis, i.e., by irradiation, though in selected cases it may beadvantageous to use acid or base catalyzed cleavage conditions.Generally irradiation is at wavelengths greater than about 340 nm,preferably at about 365 nm. The photolysis is usually conducted in thepresence of hydroxylic or protic solvents, such as aqueous, alcoholic ormixed aqueous-alcoholic or mixed aqueous-organic solvent mixtures.Alcoholic solvents frequently used include methanol and ethanol. Thephotolysis medium may also include nucleophilic scavengers such ashydrogen peroxide. Photolysis is frequently conducted at neutral orbasic pH.

[0065] This invention also provides a method of attaching a moleculewith a reactive site to a support, comprising the steps of:

[0066] (a) providing a support with a reactive site;

[0067] (b) binding a molecule to the reactive site, said first moleculecomprising a masked reactive site attached to a photolabile protectinggroup of the formula Ar—C(R₁)(R₂)—O—C(O)—, wherein:

[0068] Ar is an optionally substituted fused polycyclic aryl orheteroaromatic group or a vinylogously substituted derivative of theforegoing;

[0069] R₁ and R₂ are independently H, optionally substituted alkyl,alkenyl or alkynyl, or optionally substituted aryl or heteroaromaticgroup or a vinylogously substituted derivative of the foregoing;

[0070] to produce a derivatized support having immobilized thereon themolecule attached to the photolabile protecting group; and

[0071] (c) removing the photolabile protecting group to provide aderivatized support comprising the molecule with an unmasked reactivesite immobilized thereon.

[0072] As one of skill will recognize, the process can be repeated togenerate a compound comprising a chain of component molecules attachedto the solid support. In a “mix and match” approach, the photolabileprotecting groups may be varied at different steps in the processdepending on the ease of synthesis of the protected precursor molecule.Alternatively, photolabile protecting groups can be used in some stepsof the synthesis and chemically labile (e.g. acid or base sensitivegroups) can be used in other steps, depending for example on theavailability of the component monomers, the sensitivity of the substrateand the like. This method can also be generalized to be used inpreparing arrays of compounds, each compound being attached to adifferent and identifiable site on the support as is disclosed in U.S.Pat. Nos. 5,143,854, 5,384,261, 5,424,186 5,445,934 and copending U.S.patent application Ser. No. 08/376,963, filed Jan. 23, 1995,incorporated herein by reference for all purposes.

[0073] Thus, a related aspect of this invention provides a method offorming, from component molecules, a plurality of compounds on asupport, each compound occupying a separate predefined region of thesupport, said method comprising the steps of:

[0074] (a) activating a region of the support;

[0075] (b) binding a molecule to the region, said molecule comprising amasked reactive site linked to a photolabile protecting group of theformula Ar—C(R₁)(R₂)—O—C(O)—, wherein:

[0076] Ar is an optionally substituted fused polycyclic aryl orheteroaromatic group or a vinylogously substituted derivative of theforegoing;

[0077] R₁ and R₂ are independently H, optionally substituted alkyl,alkenyl or alkynyl, or optionally substituted aryl or heteroaromaticgroup or a vinylogously substituted derivative of the foregoing;

[0078] (c) repeating steps (a) and (b) on other regions of the supportwhereby each of said other regions has bound thereto another moleculecomprising a masked reactive site linked to the photolabile protectinggroup, wherein said another molecule may be the same or different fromthat used in step (b);

[0079] (d) removing the photolabile protecting group from one of themolecules bound to one of the regions of the support to provide a regionbearing a molecule with an unmasked reactive site;

[0080] (e) binding an additional molecule to the molecule with anunmasked reactive site;

[0081] (f) repeating steps (d) and (e) on regions of the support until adesired plurality of compounds is formed from the component molecules,each compound occupying separate regions of the support.

[0082] A related method of forming a plurality of compounds onpredefined regions of a support involves binding a molecule with areactive site protected with a chemically labile protecting group to anactivated region of the support and chemically removing the chemicallylabile protecting group to reveal the reactive site. The reactive siteis then protected with a photolabile protecting group of this invention.This process is repeated for other regions of the support with othermolecules as desired to provide a support having molecules with reactivesites protected by photolabile protecting groups on separate regions ofthe support. Reactive sites can be unmasked by removing the photolabilegroup from selected regions and coupled to additional molecules withphotolabile protecting groups as described earlier to build up arrays ofcompounds on the support. Again, in a “mix and match” approach, monomerswith chemically labile protecting groups can be attached to a reactivesite on the substrate (i.e., on the support itself when the first layerof monomers is being assembled or subsequently onto an already attachedmonomer whose reactive site has been unmasked) and these chemicallylabile protecting groups can be replaced by a photolabile protectinggroups of this invention. The replacement is accomplished by removingthe chemically labile protecting group under conditions which do notaffect any photolabile groups which may be on the support. This thenreveals an unmasked reactive site on the monomer which had carried thechemically labile protecting group and this unmasked reactive site isreacted with a reagent of the formula Ar—C(R₁)(R₂)—O—C(O)—X, where X isa leaving group. Thereby, this region of the support is protected by aphotolabile protecting group which can be selectively removed by lightdirected systems described in U.S. Pat. Nos. 5,143,854, 5,384,261,5,424,186 and 5,445,934 and further described below. This method isparticularly useful when the monomers are more readily availablecarrying chemically labile protecting groups than the photolabileprotecting groups described herein. It will be recognized that anymethod of forming a chain of compounds or an array of compounds on asupport using in at least one step a protecting group/reagent orcompound of this invention is within the scope of the methods thisinvention.

[0083] Generally, these methods involve sequential addition of monomersto build up an array of polymeric species on a support by activatingpredefined regions of a substrate or solid support and then contactingthe substrate with a protected monomer of this invention (e.g., a PYMOCprotected nucleoside or amino acid). It will be recognized that theindividual monomers can be varied from step to step. A common support isa glass or silica substrate as is used in semiconductor devices.

[0084] The predefined regions can be activated with a light source,typically shown through a screen such as a photolithographic masksimilar to the techniques used in integrated circuit fabrication. Otherregions of the support remain inactive because they are blocked by themask from illumination and remain chemically protected. Thus, a lightpattern defines which regions of the support react with a given monomer.The protected monomer reacts with the activated regions and isimmobilized therein. The protecting group is removed by photolysis andwashed off with unreacted monomer. By repeatedly activating differentsets of predefined regions and contacting different monomer solutionswith the substrate, a diverse array of polymers of known composition atdefined regions of the substrate can be prepared. Arrays of 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹² or more different polymers can be assembledon the substrate. The regions may be 1 mm² or larger, typically 10 μm²and may be as small as 1 μm². These regions are also referred to hereinas “features.”

[0085] In the preferred methods of preparing these arrays, contrastbetween features may be enhanced through the front side exposure of thesubstrate. By “front side exposure” is meant that the activation lightis incident upon the synthesis side of the substrate, contacting thesynthesis side of the substrate prior to passing through the substrate.Front side exposure reduces effects of diffraction or divergence byallowing the mask to be placed closer to the synthesis surface.Additionally, and perhaps more importantly, refractive effects from thelight passing through the substrate surface, prior to exposure of thesynthesis surface, are also reduced or eliminated by front-sideexposure. Front side exposure is described in substantial detail in U.S.patent application Ser. No. 08/634,053 filed Apr. 17, 1996, incorpratedherein by reference.

[0086] As noted previously, however, the efficiency of photolysis of thepreferred photolabile protecting groups of the present invention isimproved when such photolysis is carried out in the presence ofnucleophilic solvents, such as water or methanol. This presents a uniqueproblem where front side photolysis is used. Specifically, as the frontside of the substrate is exposed to the activation radiation, a flowcell cannot be used to maintain the desired nucleophilic environmentduring such photolysis. Accordingly, in preferred aspects,light-directed synthesis methods employing the protecting groups of thepresent invention is carried out by providing a thin aqueous film orcoating on the synthesis surface of the substrate. The presence of thisthin film or coating allows one to control the local environment on thesynthesis surface, i.e., to provide conditions that are favorable forthat synthesis. By “conditions favorable to reaction” is meantconditions that result in an improvement of reaction efficiency of agiven chemical reactant or reactants, over reactions not performed inthat environment, e.g., reaction rate, yield, or both. For example, forsynthesis methods employing the protecting groups described herein,coatings may be applied that provide a nucleophic environment which isfavorable to photolysis of the protecting group, and which therebypromotes efficient synthesis. The use of such coatings also permits thefront side exposure of the substrate surface. This method may also beperformed in reacting more than one chemical reactant, by applying bothreactants on the surface prior to coating, or by adding the secondreactant after the coating or as an element of the coating.

[0087] Generally, a thin film or coating of aqueous solution can beapplied to the synthesis surface of a substrate that is bearing theprotecting groups of the invention, e.g., that has been subjected toprevious synthesis steps. Application of the coating may be carried outby methods that are well known in the art. For example, spin-coatingmethods may be utilized where the substrate is spun during applicationof the coating material to generate a uniform coating across the surfaceof the substrate. Alternative application methods may also be used,including simple immersion, spray coating methods and the like.

[0088] Aqueous solutions for use as coating materials typically include,e.g., low molecular weight poly-alcohols, such as ethylene glycol,propylene glycol, glycerol and the like. These solutions are generallyhygrophilic and provide nucleophilic hydroxyl groups which will alsosupport the photolysis reaction. The poly-alcohols also increase theviscosity of the solution, which can be used to control the thickness ofthe coating. Higher molecular weight poly-alcohols, i.e., polyvinylalcohol, may also be used to adjust the viscosity of the coatingmaterial.

[0089] Generally, preferred substrates have relatively hydrophobicsurfaces. As such, the aqueous coating solution may also include anappropriate surfactant, e.g., from about 0.01 to about 10% v/v to permitspreading and adhesion of the film upon the substrate surface. Suchsurfactants generally include those that are well known in the art,including, e.g., Triton X-100, Tween-80, and the like. In addition topromoting the spreading and adhesion of the coating to the substrate,addition of a these non-volatile solutes within the coating solution canlimit the amount of evaporation of the film and promote its longevity.

[0090] The methods described herein may also employ component moleculescomprising a masked reactive site attached to a photolabile protectinggroup of the formula

[0091] Ar—C(R₁)(R₂)—, wherein Ar, R₁, and R₂ have the meanings ascribedearlier. In such cases, the protecting group is attached to a reactivesite that is not an amine and is removed by photolysis.

[0092] The solid substrate or solid support may be of any shape,although they preferably will be roughly spherical. The supports neednot necessarily be homogenous in size, shape or composition, althoughthe supports usually and preferably will be uniform. In someembodiments, supports that are very uniform in size may be particularlypreferred. In another embodiment, two or more distinctly differentpopulations of solid supports may be used for certain purposes.

[0093] Solid supports may consist of many materials, limited primarilyby capacity for derivatization to attach any of a number of chemicallyreactive groups and compatibility with the synthetic chemistry used toproduce the array and, in some embodiments, the methods used for tagattachment and/or synthesis. Suitable support materials typically willbe the type of material commonly used in peptide and polymer synthesisand include glass, latex, polyethylene glycol, heavily cross-linkedpolystyrene or similar polymers, gold or other colloidal metalparticles, and other materials known to those skilled in the art. Thechemically reactive groups with which such solid supports may bederivatized are those commonly used for solid phase synthesis of thepolymer and thus will be well known to those skilled in the art, i.e.,carboxyls, amines, and hydroxyls.

[0094] To improve washing efficiencies, one can employ nonporoussupports or other solid supports less porous than typical peptidesynthesis supports; however, for certain applications of the invention,quite porous beads, resins, or other supports work well and are oftenpreferable. One such support is a resin in the form of beads. Ingeneral, the bead size is in the range of 1 nm to 100 μm, but a moremassive solid support of up to 1 mm in size may sometimes be used.Particularly preferred resins include Sasrin resin (a polystyrene resinavailable from Bachem Bioscience, Switzerland); and TentaGel S AC,TentaGel PHB, or TentaGel S NH₂ resin (polystyrene-polyethylene glycolcopolymer resins available from Rappe Polymere, Tubingen, Germany).Other preferred supports are commercially available from and describedby Novabiochem, La Jolla, Calif.

[0095] In other embodiments, the solid substrate is flat, oralternatively, may take on alternative surface configurations. Forexample, the solid substrate may contain raised or depressed regions onwhich synthesis takes place. In some embodiments, the solid substratewill be chosen to provide appropriate light-absorbing characteristics.For example, the substrate may be a polymerized Langmuir Blodgett film,functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon,or any one of a variety of gels or polymers such as(poly)tetrafluorethylene, (poly)vinylidendifluoride, polystyrene,polycarbonate, or combinations thereof. Other suitable solid substratematerial will be readily apparent to those of skill in the art.Preferably, the surface of the solid substrate will contain reactivegroups, which could be carboxyl, amino, hydroxyl, thiol, or the like.More preferably, the surface will be optically transparent and will havesurface Si—OH functionalities, such as are found on silica surfaces.

[0096] The photolabile protecting groups and protected monomersdisclosed herein can also be used in bead based methods ofimmobilization of arrays of molecules on solid supports.

[0097] A general approach for bead based synthesis is described incopending application Ser. No. 07/762,522 (filed Sep. 18, 1991); Ser.No. 07/946,239 (filed Sep. 16, 1992); Ser. No. 08/146,886 (filed Nov. 2,1993); Ser. No. 07/876,792 (filed Apr. 29, 1992) and PCT/US93/04145(filed Apr. 28, 1993), Lam et al. (1991) Nature 354:82-84; PCTapplication no. 92/00091 and Houghten et al, (1991) Nature 354:84-86,each of which is incorporated herein by reference for all purposes.

[0098] Other methods of immobilization of arrays of molecules in whichthe photocleavable protecting groups of this invention can be usedinclude pin based arrays and flow channel and spotting methods.

[0099] Photocleavable arrays also can be prepared using the pin approachdeveloped by Geysen et al. for combinatorial solid-phase peptidesynthesis. A description of this method is offered by Geysen et al., J.Immunol. Meth. (1987) 102:259-274, incorporated herein by reference.

[0100] Additional methods applicable to library synthesis on a singlesubstrate are described in co-pending application Ser. No. 07/980,523,filed Nov. 20, 1992, and Ser. No. 07/796,243, filed Nov. 22, 1991,incorporated herein by reference for all purposes. In the methodsdisclosed in these applications, reagents are delivered to the substrateby either (1) flowing within a channel defined on predefined regions or(2) “spotting” on predefined regions. However, other approaches, as wellas combinations of spotting and flowing, may be employed. In eachinstance, certain activated regions of the substrate are mechanicallyseparated from other regions when the monomer solutions are delivered tothe various reaction sites. Photocleavable linkers are particularlysuitable for this technology as this delivery method may otherwiseresult in poor synthesis fidelity due to spreading, reagent dilution,inaccurate delivery, and the like. By using a photocleavable linker,rather than a conventional acid-cleavable linker, the purest materialcan be selectively cleaved from the surface for subsequent assaying orother procedures. More specifically, masks can be used when cleaving thelinker to ensure that only linker in the center of the delivery area(i.e., the area where reagent delivery is most consistent andreproducible) is cleaved. Accordingly, the material thus selectivelycleaved will be of higher purity than if the material were taken fromthe entire surface.

[0101] Typically, the molecules used in this method will be themonomeric components of complex macromolecules. These monomericcomponents can be small ligand molecules, amino acids, nucleic acids,nucleotides, nucleosides, monosaccharides and the like, thereby allowingone to synthesize arrays of complex macromolecules or polymericsequences, such as polypeptides, nucleic acids and synthetic receptors,on the solid support.

[0102] This invention discloses new nucleoside phosphoramidite monomerswith 1-pyrenylmethyloxy-carbonyl (“PYMOC”) 5′-protecting groups. Theyare photolytically cleaved under irradiation at wavelengths greater thanabout 340 nm, preferably at about 365 nm, in the presence of methanol,water, or water-solvent mixtures and/or with nucleophilic scavengerssuch as hydrogen peroxide at neutral or basic pH. The rate of photolysisis similar to that observed for the MeNPOC group. However, the yield ofPYMOC photo-removal is much higher (˜95%), so that the use of thesemonomers for photochemical synthesis of oligonucleotides leads to higherstepwise cycle yields and therefore higher-purity oligomers.

[0103] The 1-pyrenylmethyloxycarbonyl group described here can be usedfor the protection of alcohols. The photolysis of PYMOC is faster thanthat of the 1-pyrenylmethyl group, so it would also be a superiorphoto-removable protecting group for phosphates, carboxylates, amines,thiols, etc.

[0104] Other “benzylic” oxycarbonyls may have similar or betterefficiency than the PYMOC group. A general formula would be:

[0105] where Ar is an optionally substituted fused polycyclic aryl or anoptionally substituted heteroaromatic group or a vinylogouslysubstituted derivative of the foregoing;

[0106] R₁ and R₂ are independently H, optionally substituted alkyl,alkenyl or alkynyl, optionally substituted aryl, optionally substitutedheteroaromatic, or vinylogously substituted derivatives of theforegoing. Preferred embodiments are those in which Ar is a fusedpolycyclic aromatic hydrocarbon. FIG. 2 shows representative examples.Preferred substituents on the aromatic hydrocarbons would beelectron-donating groups that stabilize an incipient excited statebenzyl carbocation.

[0107] Other embodiments of the PYMOC photogroup, for example, includeat least one additional substituent at the a position, such as a methylgroup or a methoxy-substituted phenyl. These substituents will increasephotosolvolysis efficiency, and improve the selectivity for the5′-hydroxyl in the preparation of the monomer 5′-protected nucleoside.

EXAMPLES Synthesis of5′-O-PYMOC-2′-Deoxynucleoside-3′-O—(N,N-Diisopropyl)Cyanoethylphosphoramidites

[0108] All chemical reagents used were procured from commercial sources(Aldrich Chemical Co., Milwaukee, Wis. and Sigma Chemical Co.,Milawaukee, Wis.). Intermediates and products were identified by massspectrometry, ¹H-NMR, and ³¹P-NMR.

[0109] Abbreviations:

[0110] DIEA—Diethyl isopropylamine

[0111] NHS—N-hydroxysuccinimide

[0112] THF—Tetrahydrofuran

[0113] MeNPOC—methylnitropiperonyloxycarbonyl

[0114] TEA—Triethylamine

[0115] DMAP—4-Dimethylaminopyridine

[0116] Pentafluorophenyl Chloroformate

[0117] Pentafluorophenol (30 g; 163 mmol) and triethylamine (20 g, 200mmol) were combined in 200 ml dry THF, and then added dropwise to astirring solution of phosgene (20 g; 200 mmol) in 100 ml of toluene at0° C. After 2 hours, the solution was filtered and evaporated to givethe crude product as an oil, which was recrystallized from hexane toobtain 30 g (75%) pure pentafluorophenyl chloroformate.

[0118] 5′-O-(1-Pyrenylmethyl)- and 5′-O-(9-anthracenylmethyl)Oxycarbonyl-2′-deoxynucleosides

[0119] The following general procedure was used to prepare5′-PYMOC-derivatives of thymidine, N-4-isobutyryl-2′-deoxycytidine,N-7-isobutyryl-2′-deoxyadenosine, and N-4-isobutyryl-2′-deoxyguanosine;and 5′-ANMOC isobutyryl-2′-deoxyadenosine:

[0120] The base-protected nucleoside (20 mmol) was dried byco-evaporating 3 times with 50 ml dry pyridine, then dissolved in 20 mlCH₂Cl₂ and 10 ml dimethylsulfoxide (DMSO) containing 1.7 ml (21 mmol)pyridine. The resulting solution was cooled to −10° C. under argon, and5 g (20 mmol) of pentafluorophenyl chloroformate was added all at oncewith stirring. After an additional 2-3 hours stirring at −110° C., thereaction mixture was analyzed by TLC or HPLC to determine the extent ofconversion. Additional quantities of pentafluorophenyl chloroformate andpyridine (˜0.4-1.0 mmol each) were then added, as needed, until thenucleoside was completely converted to the5′-O-pentafluorophenoxycarbonyl derivative. Although isolable, at thispoint the intermediate was usually converted directly to the PYMOC orANMOC-derivative, in situ, by the addition of 1-pyrenemethanol (6 g, 26mmol) or 9-anthracenemethanol, followed by 10 ml of triethylamine and0.25 g (2 mmol) of N,N-dimethyl-aminopyridine, and stirring overnight atroom temperature. About 50-100 ml of CH₂Cl₂ was then added, and in thecase of thymidine, the pure PYMOC-derivative precipitated and could becollected by filtration. Otherwise, the solution was washed twice with5% aqueous NaHCO₃, once with saturated NaCl, dried with Na₂SO₄, andevaporated to dryness. The crude material was finally purified by flashchromatography (silica gel, 2:8 ethyl acetate-CH₂Cl₂/1-6% methanolgradient) to obtain the pure 5′-PYMOC or ANMOC nucleoside products in˜75% yield. The purity of the nucleosides was determined by HPLC,¹H-NMR, mass spectrometry and elemental analysis (CHN).

[0121]5′-O-(1-Pyrenylmethyl)oxycarbonyl-2′-deoxynucleoside-3′-O—(N,N-diisopropyl)Cyanoethylphosphoramidites

[0122] On a 12 mmol scale, the 5′-PYMOC and ANMOC nucleosides were firstdried by co-evaporation with dry pyridine, and then dissolved orsuspended in 50 ml of dry CH₂Cl₂. Then2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphorodiamidite (4.4 g; 14.5mmol) and N,N-diisopropylammonium tetrazolide (1 g; 6 mmol) were added,and the mixture was left stirring under argon overnight. The solutionwas washed twice with 10% aqueous NaHCO₃, once with saturated NaCl,dried with Na₂SO₄, and then evaporated to dryness. The crude productswere purified by flash chromatography (silica gel, eluting with a 1-5%methanol gradient in 2:8 ethyl acetate-CH₂Cl₂ containing 0.5%triethylamine) to obtain the pure phosphoramidites in ˜80% yield. Puritywas established by HPLC, ¹H, ³¹P-NMR, mass spectrometry and elementalanalysis (CHN)

[0123] Table 1 compares the efficiency of photolytic cleavage of PYMOCand ANMOC protected nucleosides to MeNPOC(methylnitropiperonyloxycarbonyl) protected nucleosides. TABLE 1Photolysis Rates: 5′-Protecting Base Group Solvent Power T_(1/2) TMeNPOC dioxane 35 mW/cm²  9 sec T PYMOC MeOH 35 mW/cm² 10 sec T PYMOC1:1 dioxane-H₂O 35 mW/cm² 10 sec T PYMOC 9:1 dioxane-MeOH 35 mW/cm² 43sec G^(ibu) MeNPOC dioxane 27 mW/cm² 11 sec G^(ibu) PYMOC MeOH 27 mW/cm²13 sec C^(ibu) MeNPOC dioxane 27 mW/cm² 12 sec C^(ibu) PYMOC MeOH 27mW/cm² 27 sec A^(ibu) MeNPOC dioxane 27 mW/cm² 12 sec A^(ibu) PYMOC MeOH27 mW/cm² 12 sec A^(ibu) ANMOC MeOH 27 mW/cm² 17 sec

[0124] Table 2 compares the coupling cycle efficiency (six cycles) ofPYMOC protected and MeNPOC protected nucleosides to ahydroxyalkylsilanated glass support using surface fluorescence analysis.TABLE 2 Stepwise Coupling Cycle Efficiencies: 1. Surface fluorescenceanalysis (“staircase” assay): 5′-Protecting Yield (6 steps) Base GroupNet Avg. Stepwise T MeNPOC 15 73 T PYMOC 56 91 dG^(ibu) MeNPOC 29 81dG^(ibu) PYMOC 61 92 dC^(ibu) MeNPOC 37 85 dC^(ibu) PYMOC 68 94 dA^(pac)MeNPOC 40 86 dA^(ibu) PYMOC 73 95 dA^(ibu) ANMOC 68 94

[0125] Table 3 compares the coupling cycle efficiency (six cycles) ofPYMOC protected and MeNPOC protected nucleosides to a solid supportusing HPLC analysis. TABLE 3 2. HPLC analysis (DOP#AF001; 3″ethenodeoxyadenosine tag): 5′-Protecting Yield (3 steps) Base Group NetAvg. Stepwise T PYMOC 92 97.2 T PYMOC 93 97.6 avg 97.4 T MeNPOC 45 77 TMeNPOC 43 75 T MeNPOC 48 78 T MeNPOC 40 74 T MeNPOC 48 78 avg 76.4

Front Side Photolysis Using PYMOC Protecting Groups

[0126] Two experiments were conducted using HO-PEG modified substrateson which 5′-PYMOC thymidine amidite had been covalently coupled in aprevious synthesis step. Striped regions of the surface were exposed at365 nm at 35 mW/cm² for 200 seconds, under each of the followingconditions: (1) dry or uncoated; (2) coated; and (3) wet (in a flow cellwith Water/MeOH).

[0127] For the coated exposures, two coatings were tested: (1) 1% TritonX-100 in H₂O; and (2) 0.2% Triton X-100 in 50% glycerol/H₂O. The coatingwas rinsed off the substrate with dry acetonitrile in a flowcell, afterthe exposure. The substrates were then stained with Fluoreprime™ amidite(Pharmacia), and scanned on a confocal laser scanner. The relativeextent of photolysis for each exposure was determined from thefluorescent intensities of each stripe. The results shown in FIG. 4 showthat both coated films greatly enhanced photolysis over the dryexposure. FIG. 4, panel A shows the fluorescent scans of uncoated or dryphotolysis (left stripe), coated photolysis using 1% Triton X-100 in H₂O(center stripe) and wet photolysis conducted in a flow cell withMethanol/H₂O (right stripe). Panel B shows uncoated or dry photolysis(left stripe), coated photolysis using 0.2% Triton X-100 in 50%glycerol/H₂O (center stripe), and wet photolysis as described above(right stripe).

[0128] The Triton-water coating showed the best performance at 95% ofthe intensity of the wet exposure (normalized at 100%), whereas theTriton/glycerol/water coating yielded 71% of the fluorescent intensity.The two dry exposures yielded 23% and 35% of the wet exposure intensity.

Hybridization Characteristics of DNA Probe Arrays Made with 5′-PYMOCPhosphoramidites

[0129] A test array comprised of 256 decanucleotides, defined by thesequence 5′-TNCNGTNCAN-3′, where N=A, C, G or T, was synthesized on anAffymetrix Array Synthesizer using 5′-PYMOC-dAiBu, dGiBu, dCiBu & Tphosphoramidites. The coupling and masking procedures used to preparethe array were the same as those described elsewhere (Pease, A. C., etal. (1994) Proc. Natl. Acad. Sci. USA 91, 5022-5026), except that thephotolysis step in each cycle was carried with methanol in contact withthe surface of the substrate. For comparison, the same array was alsosynthesized by the previously described process using5′-O-(a-methyl-6-nitropiperonyloxycarbonyl-“MeNPOC”)-nucleoside monomers(photolysis in dioxane). The array was made on a glass slide which hadbeen silanated with N,N-bis(hydroxyethyl) aminopropyltriethoxysilane asdescribed previously (Pease, A. C., et al., (1994)), and then adding tothe surface a photolysable linker,MeNPOC-hexaethyleneglycol-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite. Since oligonucleotide synthesis is more efficient withthe PYMOC-building blocks, the density of “active” synthesis sites onthe substrate was reduced prior to synthesis in order to make a bettercomparison with the less efficient array synthesis of the MeNPOCbuilding blocks. This was achieved by photo-deprotecting 90-95% of thesurface MeNPOC groups by partial photolysis (i.e., exposure to light forfour half-lives), and then capping the free hydroxyl groups with amixture of tetrazole and diethoxy-N,N-diisopropylaminophosphine. Afterdeprotecting the array in 50% ethanolic ethylenediamine for 6 hours,hybridization of a complementary fluoresein-labelled oligonucleotide“target” (5′-fluorescein-ACTGGACTGAACGGTAATGCAC-3′) was carried out at 5nM concentration in 5×-SSPE buffer (pH 7.4), in a flowcell fixed to thestage of a scanning fluorescence microscope. Hybridization to the arraywas determined by scanning the surface of the substrate to acquire asurface fluorescence image. The hybridization images that were obtainedare shown in FIGS. 5a and 5 b. The congruence of these images,demonstrates that the array fabricated with the PYMOC monomers displayedessentially the same hybridization pattern and relative intensities asthe control array made with the standard 5′-MeNPOC monomers.

[0130] The foregoing invention has been described in some detail by wayof illustration and example, for purposes of clarity and understanding.It will be obvious to one of skill in the art that changes andmodifications may be practiced within the scope of the appended claims.Therefore, it is to be understood that the above description is intendedto be illustrative and not restrictive. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to thefollowing appended claims, along with the full scope of equivalents towhich such claims are entitled.

[0131] All patents, patent applications and publications cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each individual patent, patentapplication or publication were so individually denoted.

1 2 1 10 DNA Artificial Sequence Decanucleotide used in a test array 1tncngtncan 10 2 22 DNA Artificial Sequence A complementaryfluoresein-labelled oligonucleotide “target” 2 actggactga acggtaatgc ac22

What is claimed is:
 1. A compound of the formula Ar—C(R₁)(R₂)—O—C(O)—X,wherein: Ar is an optionally substituted fused polycyclic aryl or avinylogous derivative thereof; R₁ and R₂ are H, optionally substitutedalkyl, alkenyl or alkynyl, optionally substituted aryl or optionallysubstituted heteroaromatic, or a vinylogous derivative of the foregoing;and X is a leaving group, a chemical fragment linked toAr—C(R₁)(R₂)—O—C(O)— via a heteroatom, or a solid support; provided thatwhen Ar is 1-pyrenyl and R₁ and R₂ are H, X is not linked toAr—C(R₁)(R₂)—O—C(O)— via a nitrogen atom.
 2. The compound of claim 1,wherein X is a chemical fragment selected from the group consisting ofan amino acid, a nucleic acid or analog thereof, a monosaccharide and aprotein.
 3. The compound of claim 2, wherein X is a chemical fragmentcomprising an oxygen or nitrogen atom linking X to Ar—C(R₁)(R₂)—O—C(O)—.4. The compound of claim 2, wherein X is a nucleoside or analog thereoflinked to Ar—C(R₁)(R₂)—O—C(O)— via a 3′ or 5′-OH of the nucleoside. 5.The compound of claim 4, wherein X comprises a base-protecteddeoxyribonucleoside, wherein the deoxyribonucleoside is adeoxyadenosine, a deoxycytidine, a thymidine or a deoxyguanosine.
 6. Thecompound of claim 4 wherein X comprises a base-protected ribonucleoside.7. The compound of claim 4, wherein X comprises a base-protected2′-O-methylribonucleoside.
 8. The compound of claim 4, wherein Ar isindependently 1-pyrenyl or 9-anthracenyl, R₁ is H and R₂ is H, methyl,substituted phenyl, 9-anthracenyl or 1-pyrenyl.
 9. The compound of claim8, wherein X is selected from the group consisting of base protecteddeoxyribonucleoside H-phosphonates and base protecteddeoxyribonucleoside phosphoramidites.
 10. The compound of claim 4,wherein Ar is 1-pyrenyl and R₁ and R₂ are H.
 11. A method of attaching amolecule with a reactive site to a support comprising the steps of: (a)providing a support with a reactive site; (b) binding a molecule to thereactive site, said first molecule comprising a masked reactive siteattached to a photolabile protecting group of the formulaAr—C(R₁)(R₂)—O—C(O)—, wherein: Ar is an optionally substituted fusedpolycyclic aryl or heteroaromatic group or a vinylogously substitutedderivative of the foregoing; R₁ and R₂ are independently H, optionallysubstituted alkyl, alkenyl or alkynyl, or optionally substituted aryl orheteroaromatic group or a vinylogously substituted derivative of theforegoing; to produce a derivatized support having immobilized thereonthe molecule attached to the photolabile protecting group; and (c)removing the photolabile protecting group to provide a derivatizedsupport comprising the molecule with an unmasked reactive siteimmobilized thereon.
 12. The method of claim 11, wherein the bindingstep in (b) is covalent.
 13. The method of claim 11, wherein saidremoving step is carried out in the presence of a nucleophilic solvent.14. The method of claim 13, wherein said nucleophilic solvent isselected from water, an alcohol, a water:alcohol mixture and awater:organic solvent mixture.
 15. The method of claim 14, wherein saidalcohol is selected from methanol and ethanol.
 16. The method of claim11, further comprising: (a) coupling a second molecule to the unmaskedreactive site, which second molecule comprises a second masked reactivesite attached to the photolabile protecting group to produce aderivatized support having immobilized thereon a chain of the first andsecond molecules; and (b) removing the photolabile protecting group toprovide a derivatized support with a chain of the first and secondmolecules with a second unmasked reactive site immobilized thereon. 17.The method of claim 16, further comprising repeating steps (a) and (b)of claim 10 with a succession of molecules to provide a chain ofmolecules immobilized on the support.
 18. The method of claim 17,wherein the molecules are selected from ribonucleosides,deoxyribonucleosides and 2′-O-methylribonucleosides.
 19. The method ofclaim 18, wherein the support is a glass or silica substrate.
 20. Themethod of claim 11, wherein Ar is independently 1-pyrenyl or9-anthracenyl, R₁ is H and R₂ is independently H, methyl, substitutedphenyl, 9-anthracenyl or 1-pyrenyl.
 21. The method of claim 20, whereinAr is 1-pyrenyl and R₁ and R₂ are H.
 22. The method of claim 20, whereinAr is 9-anthracenyl, R₁ is H and R₂ is H.
 23. The method of claim 18,wherein the deoxynucleosides are linked to the photolabile group via a3′ or 5′-OH.
 24. The method of claim 11, wherein the photolabile groupis removed by irradiation at a wavelength of greater than 340 nm. 25.The method of claim 24, wherein the wavelength is about 365 nm.
 26. Amethod of forming, from component molecules, a plurality of compounds ona support, each compound occupying a separate predefined region of thesupport, said method comprising the steps of: (a) activating a region ofthe support; (b) binding a molecule to the first region, said moleculecomprising a masked reactive site linked to a photolabile protectinggroup of the formula Ar—C(R₁)(R₂)—O—C(O)—, wherein: Ar is an optionallysubstituted fused polycyclic aryl or heteroaromatic group or avinylogously substituted derivative of the foregoing; R₁ and R₂ areindependently H, optionally substituted alkyl, alkenyl or alkynyl, oroptionally substituted aryl or heteroaromatic group or a vinylogouslysubstituted derivative of the foregoing; (c) repeating steps (a) and (b)on other regions of the support whereby each of said other regions hasbound thereto another molecule comprising a masked reactive site linkedto the photolabile protecting group, wherein said another molecules maybe the same or different from that used in step (b); (d) removing thephotolabile protecting group from one of the molecules bound to one ofthe regions of the support to provide a region bearing a molecule withan unmasked reactive site; (e) binding an additional molecule to themolecule with an unmasked reactive site; (f) repeating steps (d) and (e)on regions of the support until a desired plurality of compounds isformed from the component molecules, each compound occupying separateregions of the support.
 27. The method of claim 26, wherein the bindingsteps are covalent.
 28. The method of claim 27, wherein the moleculesare selected from ribonucleosides, deoxyribonucleosides and2′-O-methylribonucleosides.
 29. The method of claim 28, wherein thesupport is a glass or silica substrate.
 30. The method of claim 26,wherein Ar is independently 1-pyrenyl or 9-anthracenyl, R₁ is H and R₂is independently H, methyl, substituted phenyl, 9-anthracenyl or1-pyrenyl.
 31. The method of claim 30, wherein Ar is 1-pyrenyl and R₁and R₂ are H.
 32. The method of claim 30, wherein Ar is 9-anthracenyl,R₁ is H and R₂ is H.
 33. The method of claim 28, wherein thedeoxyribonucleosides are linked to the photolabile group via a 3′ or5′-OH.
 34. The method of claim 26, wherein the photolabile group isremoved by irradiation at a wavelength of greater than 340 nm.
 35. Themethod of claim 26, wherein the wavelength is about 365 nm.
 36. Themethod of claim 26, wherein at least 10⁶ chains are immobilized on thesupport.
 37. The method of claim 26, wherein each of the regions has anarea of between about 1 μm² and 10,000 μm².
 38. The method of claim 27,further comprising: (a) covalently binding a second molecule comprisinga masked reactive site linked to a chemically labile protecting group toa reactive site, wherein the reactive site is either on an activatedregion of the support as formed in step (a) of claim 19 or is anunmasked reactive site on a molecule on the support as formed in step(d) of claim 19; (b) replacing the chemically labile protecting groupwith the photolabile protecting group to provide a region of the supporthaving a molecule with the photolabile protecting group; and (c)repeating steps (d)-(f) of claim 19 as desired.
 39. A method ofprotecting and deprotecting a reactive group in a compound, comprising:coupling the reactive group with a second compound of the formulaAr—C(R₁)(R₂)—O—C(O)—X, wherein: Ar is an optionally substituted fusedpolycyclic aryl or heteroaromatic group or a vinylogously substitutedspecies of the foregoing; R₁ and R₂ are independently H, optionallysubstituted alkyl, alkenyl or alkynyl, or optionally substituted aryl orheteroaromatic group or a vinylogously substituted derivative of theforegoing; and X is a leaving group; provided that when Ar is 1-pyrenyland R₁ and R₂ are H, the reactive group being protected is not an amine,to provide a protected compound with its reactive group protected bylinkage to Ar—C(R₁)(R₂)—O—C(O)—; and irradiating the protected compoundto provide a deprotected compound.
 40. A method of attaching a moleculewith a reactive site to a support comprising the steps of: (a) providinga support with a reactive site; (b) binding a molecule to the reactivesite, said molecule comprising a masked reactive site attached to aphotolabile protecting group of the formula Ar—C(R₁)(R₂)—, wherein: Aris an optionally substituted fused polycyclic aryl or heteroaromaticgroup or a vinylogously substituted derivative of the foregoing; R₁ andR₂ are independently H, optionally substituted alkyl, alkenyl oralkynyl, or optionally substituted aryl or heteroaromatic group or avinylogously substituted derivative of the foregoing; to produce aderivatized support having immobilized thereon the molecule attached tothe photolabile protecting group; and (c) removing, by irradiation, thephotolabile protecting group to provide a derivatized support comprisingthe molecule with an unmasked reactive site immobilized thereon.
 41. Amethod of forming, from component molecules, a plurality of compounds ona support, each compound occupying a separate predefined region of thesupport, said method comprising the steps of: (a) activating a region ofthe support; (b) binding a molecule to the first region, said moleculecomprising a masked reactive site linked to a photolabile protectinggroup of the formula Ar—C(R₁)(R₂)—, wherein: Ar is an optionallysubstituted fused polycyclic aryl or heteroaromatic group or avinylogously substituted derivative of the foregoing; R₁ and R₂ areindependently H, optionally substituted alkyl, alkenyl or alkynyl, oroptionally substituted aryl or heteroaromatic group or a vinylogouslysubstituted derivative of the foregoing; (c) repeating steps (a) and (b)on other regions of the support whereby each of said other regions hasbound thereto another molecule comprising a masked reactive site linkedto the photolabile protecting group, wherein said another molecules maybe the same or different from that used in step (b); (d) removing, byirradiation, the photolabile protecting group from one of the moleculesbound to one of the regions of the support to provide a region bearing amolecule with an unmasked reactive site; (e) binding an additionalmolecule to the molecule with an unmasked reactive site; (f) repeatingsteps (d) and (e) on regions of the support until a desired plurality ofcompounds is formed from the component molecules, each compoundoccupying separate regions of the support.
 42. A method of protectingand deprotecting a reactive group in a compound, comprising: couplingthe reactive group with a second compound of the formula Ar—C(R₁)(R₂)—X,wherein: Ar is an optionally substituted fused polycyclic aryl orheteroaromatic group or a vinylogously substituted species of theforegoing; R₁ and R₂ are independently H, optionally substituted alkyl,alkenyl or alkynyl, or optionally substituted aryl or heteroaromaticgroup or a vinylogously substituted derivative of the foregoing; and Xis a leaving group, to provide a protected compound with its reactivegroup protected by linkage to Ar—C(R₁)(R₂)—O—C(O)—; and irradiating theprotected compound to provide a deprotected compound.
 43. A method ofattaching to a solid support a molecule with a masked reactive sitelinked to a photolabile protecting group having a formulaAr—C(R₁)(R₂)—O—C(O)—, wherein: Ar is an optionally substituted fusedpolycyclic aryl or heteroaromatic group or a vinylogously substitutedderivative of the foregoing; and R₁ and R₂ are independently H,optionally substituted alkyl, alkenyl or alkynyl, or optionallysubstituted aryl or heteroaromatic group or a vinylogously substitutedderivative of the foregoing; said method comprising the steps of: (a)activating a region of the support; (b) binding a molecule to the firstregion, said molecule comprising a masked reactive site linked to aprotecting group; (c) removing the protecting group to provide a regionbearing a molecule with an unmasked reactive site; (d) protecting theunmasked reactive site by exposing it to a reagent of the formulaAr—C(R₁)(R₂)—O—C(O)—X, wherein X is a leaving group, to provide asupport with a molecule with a masked reactive site linked to aphotolabile protecting group.
 44. A method of forming, from componentmolecules, a plurality of compounds on a support, each compoundoccupying a separate predefined region of the support, said methodcomprising the steps of: (a) activating a region of the support; (b)binding a molecule to the first region, said molecule comprising amasked reactive site linked to a protecting group; (c) repeating steps(a) and (b) on other regions of the support whereby each of said otherregions has bound thereto another molecule comprising a masked reactivesite linked to a protecting group, wherein said another molecules andprotecting groups can be the same or different 12 to each other; (d)removing the protecting group from one of the molecules bound to one ofthe regions of the support to provide a region bearing a molecule withan unmasked reactive site; (e) binding an additional molecule to themolecule with an unmasked reactive site; (f) repeating steps (d) and (e)on regions of the support, until a desired plurality of compounds isformed from the component molecules, each compound occupying separateregions of the support, with the proviso that at least one of theprotecting groups used in steps (a)-(f) is a photolabile protectinggroup of the formula Ar—C(R₁)(R₂)—O—C(O)—, wherein: Ar is an optionallysubstituted fused polycyclic aryl or heteroaromatic group or avinylogously substituted derivative of the foregoing; R₁ and R₂ areindependently H, optionally substituted alkyl, alkenyl or alkynyl, oroptionally substituted aryl or heteroaromatic group or a vinylogouslysubstituted derivative of the foregoing.
 45. The method of claim 44,wherein the binding steps are covalent.
 46. A method of making acompound of a formula Ar—C(R₁)(R₂)—O—C(O)—N, where Ar is 1-pyrenyl, R₁and R₂ are H and N is a base-protected deoxynucleoside, the methodcomprising the steps of: (a) acylating a 5′-OH of a base protecteddeoxynucleoside with pentafluorophenoxy chloroformate to provide a5′-O—C(O)-pentafluorophenoxy base-protected deoxynucleoside; (b)reacting the 5′-O—C(O)-pentafluorophenoxy base-protected deoxynucleosidewith 1-pyrenyl methyl carbinol in the presence of a base to providecompound of a formula Ar—C(R₁)(R₂)—O—C(O)—N.
 47. A method of making acompound of a formula Ar—C(R₁)(R₂)—O—C(O)—N, where Ar is 9-anthracenyl,R₁ and R₂ are H and N is a base-protected deoxynucleoside, the methodcomprising the steps of: (a) acylating a 5′-OH of a base protecteddeoxynucleoside with pentafluorophenoxy chloroformate to provide a5′-O—C(O)-pentafluorophenoxy base-protected deoxynucleoside; (b)reacting the 5′-O—C(O)-pentafluorophenoxy base-protected deoxynucleosidewith 9-anthracenyl methyl carbinol in the presence of a base to providecompound of a formula Ar—C(R₁)(R₂)—O—C(O)—N.
 48. A5′-O-pyrenylmethyloxycarbonyl base-protected deoxynucleoside made by theprocess of claim
 46. 49. A 5′-O-anthracenylmethyloxycarbonylbase-protected deoxynucleoside made by the process of claim
 47. 50. Amethod of performing chemical reactions on a surface, comprising:providing at least one chemical reactant on said surface; applying acoating to said surface, wherein said coating provides an environmentthat is favorable to reaction of said at least first chemical reactant;reacting said at least first chemical reactant.
 51. A method ofactivating a functional group on a surface of a substrate, wherein saidfunctional group is protected by a protecting group having the formulaAr—C(R₁)(R₂)—O—C(O)—X, wherein Ar is an optionally substituted fusedpolycyclic aryl or a vinylogous derivative thereof; R₁ and R2 are H,optionally substituted alkyl, alkenyl or alkynyl, optionally substitutedaryl or optionally substituted heteroaromatic, or a vinylogousderivative of the foregoing; and X is a leaving group, a chemicalfragment linked to Ar—C(R₁)(R₂)—O—C(O)— via a heteroatom, or a solidsupport; provided that when Ar is 1-pyrenyl and R₁ and R₂ are H, X isnot linked to Ar—C(R₁)(R₂)—O—C(O)— via a nitrogen atom, the methodcomprising: applying a nucleophilic coating to said surface; andexposing said surface to light to remove said protecting group.
 52. Themethod of claim 51, wherein said nucleophilic coating comprises asurfactant.
 53. The method of claim 51, wherein said surfactant ispresent at a concentration of from about 0.01% to about 10% v/v.
 54. Themethod of claim 51, wherein said coating comprises a poly-alcohol. 55.The method of claim 54, wherein said poly-alcohol is selected fromglycerol, ethylene glycol, and propylene glycol.
 56. The method of claim51, wherein said coating is spin coated onto said substrate surface. 57.The method of claim 39, wherein Ar is independently 1-pyrenyl or9-anthracenyl, R₁ is hydrogen and R₂ is independently hydrogen, methyl,substituted phenyl, 9-anthracenyl or 1-pyrenyl.
 58. The method of claim42, wherein Ar is independently 1-pyrenyl or 9-anthracenyl, R₁ ishydrogen and R₂ is independently hydrogen, methyl, substituted phenyl,9-anthracenyl or 1-pyrenyl.
 59. The method of claim 58, wherein Ar is1-pyrenyl and R₁ and R₂ are hydrogen.
 60. The method of claim 58,wherein Ar is 9-anthracenyl, R₁ is hydrogen and R₂ is hydrogen.
 61. Themethod of claim 42, wherein the compound is a ribonucleoside,deoxyribonucleoside or 2′-O-methylribonucleoside.
 62. The method ofclaim 61, wherein the deoxyribonucleoside is linked to theAr—C(R₁)(R₂)—O—C(O)— group via a 3′ or 5′-OH.
 63. The method of claim42, wherein the Ar—C(R₁)(R₂)—O—C(O)— group is removed by irradiationwith light
 64. The method of claim 63, wherein the wavelength is greaterthan 340 nm.
 65. The method of claim 64, wherein the wavelength is about365 nm.